Summary of Work: Identification of Bioactive Isoforms of p53 Protein P53 protein is involved in regulation of the cell cycle, apoptosis and cell differentiation. While the rise and fall of total p53 levels are often correlated with function, it does not reflect the dynamic changes in phosphorylation state. The purpose of this project is to study how changes in p53 phosphorylation relate to molecular function. Several approaches are being used to examine the relationship between p53 phosphorylation and function. 1) 2D PAGE is being used to separate p53 phosphoisoforms and note proportional changes and appearance of new species in response to model DNA damaging agents. Individual isoforms will be related to p53-mediated cellular responses to identify bioactive isoforms. 2) Production of recombinant p53 from the baculovirus expression system is being used to identify normal phosphorylated and hyperphosphorylated p53 digestion fragments by mass spectral (MS) analysis in collaboration with LSB. MS analysis will also be similarly applied to purified p53 from normal cells in response to DNA damaging agents. 3) Stable p53 in normal human mammary epithelial cells (HMEC) is being studied for response DNA damaging agents to determine differences in response from cells expressing labile p53. Variations in microcellular environment will be examined to account for variation in p53 stability, degradation and phosphoisoform turnover. 4) Tumor cell lines expressing wild type p53 will be compared to normal human diploid cells for differences in response to cell cycle arrest and apoptosis and which p53 phosphoisoform expression pattern is correlated with each outcome. 5) Differential display RT PCR analysis is being developed to examine gene expression from genotypically identical cells expressing wild versus mutant p53 protein. Thus, ongoing and collaborative work will attempt to link specific biological activities to individual p53 isoforms.